MDM2 and its homolog MDMX are important regulators of the p53 tumor suppressor. Knockout experiments showed that both are essential for viability of mouse embryos. In human tumor cells, MDMX knockdown inhibits tumor xenograft formation, whereas MDMX overexpression increases tumor resistance to chemotherapy. Unlike MDM2, MDMX does not have E3 ligase activity and its mechanism of p53 regulation is poorly understood. We found that MDMX is an integral part of signaling pathways that activate p53. DNA damage induces MDMX phosphorylation by ATM and Chk2, leading to its degradation by MDM2. Oncogenic and nucleolar stress also induce MDMX degradation by ARF and L11 regulation of MDM2. Therefore, accelerated MDMX degradation is a common mechanism for p53 activation. We found that 14-3-3 proteins regulate MDMX degradation after DNA damage by binding to a Chk2 phosphorylation site (S367) on MDMX. We have also identified CK11 as an MDMX binding partner that phosphorylates MDMX S289 and enhances the ability of MDMX to inhibit p53. We hypothesize that signaling to p53 is in part mediated through regulation of MDMX phosphorylation and degradation. The following experiments are proposed to further study the function and regulation of MDMX during stress response. (1) Determine the mechanism of p53 inhibition by MDMX. (2) Investigate the regulation of MDMX-p53 interaction by Chk2 and CK1?. (3) Investigate the regulation of MDMX degradation. (4) Test the in vivo functions of MDMX interactions with 14-3-3 and CK1?. These experiments should lead to a better understanding of the mechanisms that regulate MDMX and may identify novel strategy for targeting MDMX in human cancer. PUBLIC HEALTH RELEVANCE: The proposal will study the regulation of the mdmx oncoprotein and the mechanism of p53 inactivation by mdmx. The experiments are based on recent findings on the regulation of mdmx degradation by phosphorylation and interactions with kinases and ribosomal proteins. The experiments will also use mouse models to test the physiological functions of mdmx modifications. Understanding the mechanisms of mdmx regulation may lead to new strategies to activate p53 in cancer cells.